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J W Fawcett

Publications and source records attributed to J W Fawcett.

At least 19 recordsLinked to original sources
PubMed1999

Comparing astrocytic cell lines that are inhibitory or permissive for axon growth: the major axon-inhibitory proteoglycan is NG2.

Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenitors (O2A cells) can all produce molecules that inhibit axon regeneration. We have shown previously that inhibition of axon growth by astrocytes involves proteoglycans. To identify inhibitory mechanisms, we created astrocyte cell lines that are permissive or nonpermissive and showed that nonpermissive cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We have now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PGs in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was tested using a function-blocking antibody. This allowed increased growth of dorsal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increased the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 cells; CS-56 antibody had a similar effect. Inhibitory fractions of conditioned medium contained NG2 coupled to CS glycosaminoglycan chains, whereas noninhibitory fractions contained NG2 without CS chains. Enzyme preparations that facilitated axon growth in Neu7 cultures were shown to either degrade the NG2 core protein or remove CS chains. Versican is present as patches on Neu7 monolayers, but DRG axons do not avoid these patches. Therefore, NG2 appears to be the major axon-inhibitory factor made by Neu7 astrocytes. In the CNS, NG2 is expressed by O2A cells, which react rapidly after injury to produce a dense NG2-rich network, and by some reactive astrocytes. Our results suggest that NG2 may be a major obstacle to axon regeneration.

Animals↗
PubMed1999

Regulation of fibronectin alternative splicing during peripheral nerve repair.

Wallerian degeneration following peripheral nerve injury is associated with increased production of fibronectin and other extracellular matrix molecules that are thought to enhance repair. We have shown previously that alternative splicing of the mRNA for fibronectin also changes following sciatic nerve lesions so as to reexpress forms of mRNA seen during embryogenesis. In the present study, we have examined the role of the regenerating axons in the regulation of this splicing. We have compared the patterns of fibronectin mRNA splicing seen in sciatic nerve development with that seen in cut nerves (that do not regenerate), crushed nerves (that regenerate successfully), and Schwann cells cultured in forskolin so as to mimic axonal signals. By using a reverse transcriptase polymerase chain reaction assay to examine all three regions of fibronectin mRNA splicing in a quantitative manner, we found that embryonic patterns of fibronectin mRNA splicing appear rapidly following injury and are not then altered by reestablishment of axons in the nerve. In addition, we found that forskolin has no effect on fibronectin mRNA splicing in cultured cells. We conclude that axonal signals do not regulate the pattern of fibronectin alternative splicing in peripheral nerve repair.

Alternative Splicing↗
PubMed1999

Delayed implantation of nigral grafts improves survival of dopamine neurones and rate of functional recovery.

In order to test the hypothesis that poor survival of dopaminergic neurones in nigral transplants may be due, at least in part, to acute toxic changes in the host striatum within the first hour after injury, we experimentally evaluated the consequences of imposing a brief delay (20 min, 1 or 3 h) between positioning the injection cannula and extruding the graft tissue. A delay of as little as 1 h resulted in a three-fold increase in survival of dopamine neurones in the grafts and a more rapid abolition of amphetamine-induced rotational asymmetry in the host animals. These results suggest that acute but rapidly resolving changes in the host striatal environment induced by the implantation procedure itself can have a significantly deleterious effect on the survival of embryonic nigral grafts.

Amphetamine↗
PubMed1999

A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival and fiber outgrowth from embryonic nigral neurons grafted to the striatum and to the lesioned substantia nigra.

We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.

Animals↗
PubMed1999

Addition of fresh blood to intrastriatal grafts of embryonic mesencephalon into the hemiparkinsonian rat does not impair the survival of grafted dopaminergic neurones.

Cell transplantation therapy for Parkinson's patients, although seen to bring benefit to some patients during first clinical trials, remains impracticable on a large scale, in part because of the poor survival of the dopaminergic neurones transplanted. The loss of dopaminergic neurones occurs rapidly over the first 1-2 days after transplantation, in response to factors intrinsic to the host brain. Here we investigated whether contamination of the grafted cell suspension with blood during the transplantation procedure may be one factor responsible for the poor survival of DA neurons within the graft, possibly through factors such as free iron or complement. 6-Hydroxydopamine lesioned rats were grafted with 2 microl suspension of dissociated E14 ventral mesencephalon to which 1 microl blood or 1 microl grafting medium was added. After 6 weeks, there was no significant difference in the number of surviving DA neurones in the two groups. We conclude that contamination of grafts with blood is not a major factor responsible for the extensive death of dopaminergic neurones within them.

Animals↗
PubMed1999

Enhanced axonal regeneration following combined demyelination plus schwann cell transplantation therapy in the injured adult spinal cord.

We have treated spinal cord injured rats with demyelination plus Schwann cell transplantation and assessed neurite outgrowth in a quantifiable model of axonal regeneration. Axonal injuries of differing severity were induced in the dorsal funiculus of adult rats using a micromanipulator-controlled Scouten knife. Demyelinated regions were produced so as to overlap with the injury site by the injection of galactocerebroside antibodies plus complement one segment cranial to the axonal injury site. Schwann cells were isolated from the sciatic nerve, expanded in vitro, and transplanted into the injury site 1 day later. Animals were killed after an additional 7 days. Schwann cells were evenly distributed throughout the region of demyelination, which extended 6-7 mm cranial to the axonal injury site. The severity of axonal injury was quantified by counting degenerate axons in transverse resin sections. The degree of axonal regeneration was assessed by an electron microscopic analysis of growth cone frequency and distribution relative to the site of axonal injury. Quantification of growth cones at a distance from the site of axonal injury indicated a strong linear relationship (P < 0.001) between the number of growth cones and the number of severed axons; the ratio of growth cones to severed axons was increased by 26.5% in demyelinated plus transplanted animals compared to demyelinated animals without a transplant. Furthermore, only the demyelinated plus transplanted animals contained growth cones associated with myelin in white matter immediately outside of the region of complete demyelination. Growth cones were absent in transplanted-only animals at a distance from the site of axonal injury. These findings indicate that combined demyelination plus Schwann cell transplantation therapy enhances axonal regeneration following injury and suggests that growth cones are able to overcome myelin-associated inhibitors of neurite outgrowth in the presence of trophic support.

Age Factors↗
PubMed1999

N-Cadherin inhibits Schwann cell migration on astrocytes.

Astrocytes exclude Schwann cells (SCs) from the central nervous system (CNS) at peripheral nerve entry zones and restrict their migration after transplantation into the CNS. We have modeled the interactions between SCs, astrocytes, and fibroblasts in vitro. Astrocytes and SCs in vitro form separate territories, with sharp boundaries between them. SCs migrate poorly when placed on astrocyte monolayers, but migrate well on various other surfaces such as laminin (LN) and skin fibroblasts. Interactions between individual SCs and astrocytes result in long-lasting adhesive contacts during which the SC is unable to migrate away from the astrocyte. In contrast, SC interactions with fibroblasts are much shorter with less arrest of migration. SCs adhere strongly to astrocytes and other SCs, but less well to substrates that promote migration, such as LN and fibroblasts. SC-astrocyte and SC-SC adhesion is mediated by the calcium-dependent cell adhesion molecule N-cadherin. Inhibition of N-cadherin function by calcium withdrawal, peptides containing the classical cadherin cell adhesion recognition sequence His-Ala-Val, or antibodies directed against this sequence inhibit SC adhesion and increase SC migration on astrocytes. We suggest that N-cadherin-mediated adhesion to astrocytes inhibits the widespread migration of SCs in CNS tissue.

Amino Acid Sequence↗
PubMed1999

Dehydroepiandrosterone antagonizes the neurotoxic effects of corticosterone and translocation of stress-activated protein kinase 3 in hippocampal primary cultures.

Glucocorticoids are toxic to hippocampal neurons. We report here that the steroid dehydroepiandrosterone protects neurons of primary hippocampal cultures against the toxic effects of corticosterone. Corticosterone (20-500 nM) added for 24h to primary cultures of embryonic day 18 rat hippocampus resulted in significant neurotoxicity. Dissociated cells were grown for at least 10 days, initially in serum-containing medium, but serum was removed before adding steroids for 24 h. Neurotoxicity was measured by counting the number of cells stained either for beta-tubulin III or glial fibrillary acidic protein. Corticosterone-induced toxicity was prevented by co-treatment of the cultures with dehydroepiandrosterone (20-500 nM). Dehydroepiandrosterone on its own had little effect, though the highest concentration used (500 nM) was mildly toxic. Immunohistochemical studies on the nuclear translocation of a range of stress-activated protein kinases showed that stress-activated protein kinases 1, 2, 3 and 4 were all translocated by 10 min exposure to corticosterone (100 nM). Dehydroepiandrosterone (100 nM) attenuated the translocation of stress-activated protein kinase 3, but not the others. These experiments show that dehydroepiandrosterone has potent anti-glucocorticoid actions on the brain, and can protect hippocampal neurons from glucocorticoid-induced neurotoxicity. This protective action may involve stress-activated protein kinase 3-related intracellular pathways, though direct evidence for this has still to be obtained.

Animals↗
PubMed1999

The glial scar and central nervous system repair.

Damage to the central nervous system (CNS) results in a glial reaction, leading eventually to the formation of a glial scar. In this environment, axon regeneration fails, and remyelination may also be unsuccessful. The glial reaction to injury recruits microglia, oligodendrocyte precursors, meningeal cells, astrocytes and stem cells. Damaged CNS also contains oligodendrocytes and myelin debris. Most of these cell types produce molecules that have been shown to be inhibitory to axon regeneration. Oligodendrocytes produce NI250, myelin-associated glycoprotein (MAG), and tenascin-R, oligodendrocyte precursors produce NG2 DSD-1/phosphacan and versican, astrocytes produce tenascin, brevican, and neurocan, and can be stimulated to produce NG2, meningeal cells produce NG2 and other proteoglycans, and activated microglia produce free radicals, nitric oxide, and arachidonic acid derivatives. Many of these molecules must participate in rendering the damaged CNS inhibitory for axon regeneration. Demyelinated plaques in multiple sclerosis consists mostly of scar-type astrocytes and naked axons. The extent to which the astrocytosis is responsible for blocking remyelination is not established, but astrocytes inhibit the migration of both oligodendrocyte precursors and Schwann cells which must restrict their access to demyelinated axons.

Animals↗
PubMed1999

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective.

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.

Animals↗
PubMed1999

Dopamine cells in nigral grafts differentiate prior to implantation.

The yield of surviving dopamine cells in nigral grafts is typically low. It is unclear whether the dopamine neurons that do survive are postmitotic at the time of implantation, or are precursor cells that differentiate into dopamine neurons following transplantation in the host brain. We have therefore compared the survival of dopamine neurons in grafts that have been labelled with BrdU at different times prior to or following implantation in order to identify those cells that undergo final cell division at each stage of the procedure. Seven groups of rats were prepared with unilateral nigrostriatal lesions. Three groups received nigral grafts derived from E14 embryos labelled with BrdU in utero on either E12, E13 or E14 days of embryonic age (the E14 injection made 2 h prior to preparation of the graft cell suspension). Three further groups received nigral grafts from untreated E14 embryos, and then dividing cells within the grafts were labelled by injection of BrdU into the host lateral ventricle, 2 h, 1 day or 2 days after implantation (equivalent to E14, E15 and E16 days of embryonic age). The control group received standard (unlabelled) E14 grafts. Five weeks after the transplantation surgery, the host brains were processed using double immunohistochemical techniques to detect tyrosine hydroxylase (TH)-positive neurons which had incorporated BrdU. In the grafts labelled with BrdU prior to implantation, there was an increasing proportion of double-labelled cells (out of the total TH-positive cells surviving in the grafts) with birth dates on E12, E13 and E14 (1%, 12% and 10% per day, respectively). By contrast, grafts labelled following implantation, although containing many dividing neurons, had very few of these BrdU-labelled cells expressing a dopaminergic phenotype; < 1% surviving TH-positive cells were double-labelled from the 2 h post-transplant injection, and < 0.1% from each subsequent injection. This suggests not only that the great majority of TH-positive neurons in nigral grafts were already differentiated at the time of implantation, but also that transplantation of E14 ventral mesencephalic tissue either kills dopaminergic precursors or (more likely in our opinion) prevents their differentiation into a dopaminergic phenotype. Precursor cells that would differentiate into dopaminergic neurons beyond E14 if left in situ in the intact ventral mesencephalon do not readily differentiate into mature dopamine neurons following transplantation. If we are to enhance yields of functional dopamine-rich transplants, then we must identify strategies both to protect predifferentiated dopamine neurons in the grafts and to promote differentiation of a dopaminergic phenotype in precursor cells that continue to divide within the grafts following transplantation into an adult host environment.

Animals↗
PubMed1998

Spinal cord repair: from experimental models to human application.

Axon regeneration fails in the CNS because the glial environment is inhibitory, and because the regenerative response of CNS is poor. Regeneration can therefore be induced by removing the inhibitory effect of CNS glial molecules, by increasing the regenerative in animal models of spinal cord injury has recently been achieved by several strategies that apply these principles. The successful techniques have been to block inhibitory molecules made by astrocytes, to implant peripheral nerve grafts embedded in a bFGF-containing fibrin gel, to implant olfactory ensheathing cells, to graft embryonic spinal cord tissue, and to implant trophic factor-secreting fibroblasts. The next challenge is to prepare to apply these types of treatment to human patients with spinal cord injuries.

Animals↗
PubMed1998

Cytokine-induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth.

Repair of demyelination in the CNS requires that oligodendrocyte precursors (OPs) migrate, divide and then myelinate. Repair of axon damage requires axonal regeneration. Limited remyelination and axon regeneration occurs soon after injury, but usually ceases in a few days. In vivo and in vitro experiments have shown that astrocytic environments are not very permissive for migration of OPs or for axonal re-growth. Yet remyelination and axon sprouting early after injury occurs in association with astrocytes, while later astrocytes can exclude remyelination and prevent axon regeneration. A large and changing cast of cytokines are released following CNS injury, so we investigated whether some of these alone or in combination can affect the ability of astrocytes to support migration of OPs and neuritic outgrowth. Interleukin (IL) 1alpha, tumour necrosis factor alpha, transforming growth factor (TGF) beta, basic fibroblast growth factor (bFGF), platelet-derived growth factor and epidermal growth factor alone exerted little or no effect on migration of OPs on astrocytes, whereas interferon (IFN) gamma was inhibitory. The combination of IL-1alpha + bFGF was found to be pro-migratory, and this effect could be neutralized by TGFbeta. We also examined neuritic outgrowth from dorsal root ganglion explants in three-dimensional astrocyte cultures treated with cytokines and found that IL-1alpha + bFGF greatly increased axon outgrowth and that this effect could be blocked by TGFbeta and IFNgamma. All these effects were absent or much smaller when OP migration or axon growth was tested on laminin, so the main effect of the cytokines was via astrocytes. The cytokine effects did not correlate with expression on astrocytes of laminin, fibronectin, tenascin, chondroitin sulphate proteoglycan, N-cadherin, polysialyated NCAM (PSA-NCAM), tissue plasminogen activator (tPA) or urokinase (uPA).

Animals↗
PubMed1998

Increased axon growth through astrocyte cell lines transfected with urokinase.

The ability of cells to migrate through tissues depends on their production of a variety of proteases, and the same may be true of growth cones. Urokinase (plasminogen activator) regulates much of the extracellular proteolytic activity, by activating other proteases and as a result of its own proteolytic activity. In order to evaluate the potential role of urokinase as a promoter of axon growth, we have used a plasmid expressing urokinase under a cytomegalovirus promoter to transfect an astrocyte cell line, Neu7, which we have previously shown to provide a poor environment for axon regeneration. Five transfected lines all showed greatly increased ability to promote axon regeneration in both monolayer and three-dimensional cultures. The critical change in the transfected cells was largely within the extracellular matrix, since extracellular matrix laid down by urokinase-secreting cells was more permissive to axon growth than matrix from the parent Neu7 line. The effect was due to urokinase since treatment of the transfected cells with the urokinase inhibitors B623 and B428 rendered both the cells and their matrix much less permissive to axon growth, but did not require plasminogen, since it was blocked neither by serum-free medium nor by plasmin inhibitors.

Animals↗
PubMed1997

Proteoglycans provide neurite guidance at an astrocyte boundary.

Astrocytes in the developing brain direct neurites through their synthesis of cell surface and extracellular matrix molecules. We introduce a novel culture system to identify and examine the guidance properties of astrocyte-derived molecules. The permissive A7 and nonpermissive Neu7 cell lines were co-cultured to form an A7/Neu7 monolayer. Neurites extended on A7 cells but avoided Neu7 cells and instead stopped or turned at the A7/Neu7 Interface. Enzymatic treatment with trypsin and hyaluronic acid increased neurite extension, but neither altered the boundary. Only, removal of keratan and chondroitin sulfate residues reduced the guidance capacity of the A7/Neu7 boundary. Since no treatment individually abolished the boundary, neurite guidance appears to be due to a combination of factors. The A7/Neu7 astrocyte substrate demonstrates the functional role for KSPGs and CSPGs, but more interestingly, suggests that simply increasing the capacity of a substrate to permit neurite outgrowth does not necessarily eliminate or even reduce its guidance properties.

Animals↗
PubMed1997

Astrocytic and neuronal factors affecting axon regeneration in the damaged central nervous system.

Whether an axon will regenerate after it is cut depends on the balance between the intrinisic ability of the axon to regrow and the permissiveness of the environment surrounding it. The permissiveness of the environment is determined by the glial cells at the site of injury, and in the CNS both oligodendrocytes and astrocytes produce inhibitory molecules. Neurones differ greatly in the ability of their axons to regrow following axotomy. This intrinsic growth ability is greater in embryonic than adult neurones, varies from one neuronal type to another, depends on whether the axon is cut close to or far from the cell body, and can be increased by appropriate neurotrophic molecules.

Astrocytes↗
PubMed1996

GDNF enhances dopaminergic cell survival and fibre outgrowth in embryonic nigral grafts.

Two groups of rats with unilateral 6OHDA lesions received either intrastriatal suspension grafts of embryonic ventral mesencephalon or sham grafts. Three subgroups of each of these received intrastriatal infusions of 1000 ng or 500 ng glial cell-line derived trophic factor (GDNF) or vehicle alone for 10 consecutive days. There was a highly significant dose-dependent effect of GDNF both on the number of TH-positive cells surviving in the grafts and on the density of fibre outgrowth. All grafted rats showed rapid compensation of amphetamine-induced rotation compared with rats with sham grafts. GDNF may provide a powerful tool to enhance the survival and maturation of dopaminergic neurones within mesencephalic transplants.

Animals↗